We describe multiphoton imaging with sample-temperature control to monitor animal cells and cells of intact plants during freezing, thawing and heating processes based on autofluorescence intensity and lifetime. The sample temperature can be set with a heating and freezing stage to any value in the range between liquid nitrogen temperature (−196 °C; 77 K) and +600 °C (873 K) and changed with adjustable heating/freezing rates between 0.01 K/min and 150 K/min. Multiphoton imaging is realized with near-infrared femtosecond-laser excitation with different setups employing different laser sources. To illustrate the capabilities, imaging of animal cell samples with and without a cryoprotectant during freezing at cooling rates is presented. Lowering the temperature led to a significant increase of the intracellular fluorescence intensity and modifications. Fluorescence lifetime imaging indicated an increase of the mean lifetime with decreasing temperature. Furthermore, to illustrate imaging of plant samples, Arabidopsis thaliana leaves were employed. The measurements revealed thermally-induced changes of fluorescence lifetime and intensity as well as morphological alterations in the distribution of chloroplasts. The measurements illustrate the general usefulness of multiphoton imaging to investigate freezing and thawing effects on animal and plant cells even at temperatures commonly used for cryopreservation.
Nonlinear contrast methods such as two-photon excited autofluorescence (in combination with fluorescence lifetime imaging) and second-harmonic generation have been combined with a further multiphoton contrast mechanism called coherent anti-Stokes Raman scattering. We describe the principle and the instrumentation for its implementation in tomographs for dermatological applications and present multimodal optical skin biopsies.
Dysfunctions and dystrophies severely affect the cornea’s function. In point of fact, cornea diseases are the second major cause of blindness worldwide. Corneal diagnosis in clinical practice heavily relies on imaging techniques such as slit lamp microscopy, confocal microscopy, or optical coherence tomography. However, these fail to provide information on the cell’s metabolic state or the structural organization of the corneal stroma. With two-photon microscopy and fluorescence lifetime imaging this information can be obtained. Therefore, corneal pathology diagnosis may be improved. The feasibility of corneal characterization by two-photon imaging has been demonstrated in ex vivo samples and in vivo animal models. In this chapter, we report on the use of two multiphoton microscopy instruments for imaging the human cornea: a 5D multiphoton laser scanning microscope and the multiphoton tomograph MPTflex. Human corneas unsuitable for transplantation but otherwise normal and pathological samples obtained after surgery were imaged and characterized based on their autofluorescence and second-harmonic generation signals. Two possible clinical applications of two-photon microscopy are discussed: (i) the assessment of tissue viability before corneal transplantation and (ii) the differential diagnosis of corneal pathologies, further demonstrating the advantages of this imaging modality for corneal diagnosis.